Phosphoinositide 3-kinases (hereafter, “PI3Ks”) are enzymes that phosphorylate the 3-hydroxyl position of the inositol ring of phosphoinositides (“PIs”), and are involved in diverse cellular events such as cell migration, cell proliferation, oncogenic transformation, cell survival, signal transduction, and intracellular trafficking of proteins. In yeast (S. cerevisiae), Vps34 (“vacuolar protein sorting 34”) encodes a PI 3-kinase gene product that mediates the active diversion of proteins from the secretory pathway to vacuoles; mammals have a corresponding family of PI3-kinases, including three classes of PI3Ks, with a variety of isoforms and types within. The closest human homolog of yeast Vps34 is an 887 residue protein called PI3Kclass3 (“PI3KC3”) (also referred to herein as “hVps34,” for human Vps34), which shares about 37% sequence identity with the yeast protein over its full length (Volinia et al. (1995) EMBO J. 14(14): 3339).
hVps34 is the enzymatic component of a multiprotein complex that includes a ser/thr kinase called Vps15p, and proautophagic tumor suppressors Beclin1/Atg6, and UVRAG, and Bif-1 in mammals (Vps15, Atg6, and Atg 14 in yeast) (Mari et al. (2007) Cell Biol. 9:1125). Of the three classes of PI3 kinase this has the most restricted substrate specificity, being strictly limited to PtdIns. Like class IA PI3-kinases, PI3KC3s (e.g., hVps34) play a well recognized role in the regulation of S6K1, and hence in nutrient-sensing.
The Vps34 gene product (Vps34p) is an enzyme required for protein sorting to the lysosome-like vacuole of the yeast, and appears to regulate intracellular protein trafficking decisions. Vps34p shares significant sequence similarity with the catalytic subunit of bovine phosphatidylinositol (PI) 3-kinase (the p110 subunit), which is known to interact with activated cell surface receptor tyrosine kinases. Yeast strains deleted for the Vps34 gene or carrying Vps34 point mutations lacked detectable PI 3-kinase activity and exhibited severe defects in vacuolar protein sorting. Overexpression of Vps34p resulted in an increase in PI 3-kinase activity, and this activity was specifically precipitated with antisera to Vps34p (Schu et al. (1993) Science 260 (5104): 88). hVps34 is an integral part of the autophagy process, or eukaryotic cell mechanism for cytoplasmic renewal. Autophagy is a cellular catabolic degradation response to starvation or stress whereby cellular proteins, organelles, and cytoplasm are engulfed, digested, and recycled to sustain cellular metabolism. Autophagy is characterized by the engulfment of cytoplasmic material into specialized double-membrane vesicles known as autophagosomes. The degradation of cellular cytoplasmic components helps eukaryotic cells jettison defective organelles and protein complexes (Yorimitsu et al (2005) Cell Death Differ. 12:1542). Nonselective autophagy can be initiated by starvation, allowing cells to convert organelles and proteins into nutrients for survival. Autophagy can also be employed to kill cells, in addition to or instead of apoptosis, for instance (Neufeld et al (2008) Autophagy).
Parodoxically, certain pathologies feature autophagy processes, such as tumorigenesis, aging, and neurodegeneration (Huang et al. (2007) Cell Cycle 6:1837). Cell death resulting from progressive cellular consumption has been attributed to unmitigated autophagy (Baehrecke et al. (2005) Nature Rev. Mol. Cell. Biol. 6:505). Autophagy is thought to prolong the survival of tumor cells defective in apoptosis, e.g., protecting them from metabolic stress. Inhibiting autophagy, and thereby sensitizing cells (e.g., apoptosis-resistant cells) to metabolic stress represents a promising tumor therapy regimen (Mathew et al. (2007) Nature Reviews 7:961). Known inhibitors of autophagy, including Wortmannin and 3-methyladenine, target and inhibit hVps34 (Petiot et al. (2000) J. Biol. Chem. 275:992).
Consistent with the autophagy and cancer link is recognition in the field that constitutive activation of PI3Ks is responsible for at least ovarian, head and neck, urinary tract, cervical and small cell lung cancers. PI3K signaling can be attenuated by the phosphatase activity of the tumor suppressor PTEN (phosphatase and tensin homologue detected in chromosome 10), which is absent in a number of human cancers. Inhibiting PI3K arrests a major cancer cell survival signaling pathway and overcomes the absence of tumor suppressor PTEN, providing antitumor activity and increased tumor sensitivity to a wide variety of drugs (Stein et al. (2001) Endocrine-Related Cancer 8:237).